GxcDD, a putative RacGEF, is involved in Dictyostelium development
- Subhanjan Mondal†1,
- Dhamodharan Neelamegan†1, 2,
- Francisco Rivero1 and
- Angelika A Noegel†1Email author
© Mondal et al; licensee BioMed Central Ltd. 2007
Received: 18 December 2006
Accepted: 20 June 2007
Published: 20 June 2007
Rho subfamily GTPases are implicated in a large number of actin-related processes. They shuttle from an inactive GDP-bound form to an active GTP-bound form. This reaction is catalysed by Guanine nucleotide exchange factor (GEFs). GTPase activating proteins (GAPs) help the GTPase return to the inactive GDP-bound form. The social amoeba Dictyostelium discoideum lacks a Rho or Cdc42 ortholog but has several Rac related GTPases. Compared to our understanding of the downstream effects of Racs our understanding of upstream mechanisms that activate Rac GTPases is relatively poor.
We report on GxcDD (G uanine ex change factor for Rac GTPases), a Dictyostelium RacGEF. GxcDD is a 180-kDa multidomain protein containing a type 3 CH domain, two IQ motifs, three PH domains, a RhoGEF domain and an ArfGAP domain. Inactivation of the gene results in defective streaming during development under different conditions and a delay in developmental timing. The characterization of single domains revealed that the CH domain of GxcDD functions as a membrane association domain, the RhoGEF domain can physically interact with a subset of Rac GTPases, and the ArfGAP-PH tandem accumulates in cortical regions of the cell and on phagosomes. Our results also suggest that a conformational change may be required for activation of GxcDD, which would be important for its downstream signaling.
The data indicate that GxcDD is involved in proper streaming and development. We propose that GxcDD is not only a component of the Rac signaling pathway in Dictyostelium, but is also involved in integrating different signals. We provide evidence for a Calponin Homology domain acting as a membrane association domain. GxcDD can bind to several Rac GTPases, but its function as a nucleotide exchange factor needs to be studied further.
Rho GTPases are small monomeric GTPases of the Ras superfamily. Like any other GTPase Rho GTPases act as binary molecular switches cycling between a GTP-bound active and a GDP-bound inactive form. Guanine nucleotide exchange factors (GEFs) catalyze the activation reaction, and GTPase activating proteins (GAPs) convert the active to an inactive form. Further regulators, guanine nucleotide dissociation inhibitors (GDIs), block spontaneous activation and regulate cycling between membrane and cytosol. When activated, Rho GTPases undergo a conformational change enabling them to interact with their effector molecules and transduce signals for downstream events. Rho GTPases have been implicated in a large number of actin-related processes like motility, adhesion, morphogenesis, membrane trafficking and cytokinesis [1, 2].
The human genome codes for 21 Rho GTPases and functions of most of them are only poorly understood. Of these, three, namely RhoA, Rac1 and Cdc42 are more extensively studied. RhoA generates myosin-based contractility and formation of adhesion complexes; Rac1 and Cdc42 are primarily involved in formation of protrusive structures, Rac1 regulates formation of lamellipodia and Cdc42 regulates filopodia formation and establishment of cell polarity [1, 2].
Sequencing of the genome of the social amoeba Dictyostelium discoideum revealed the presence of 18 Rac related GTPases, whereas a typical Rho or Cdc42 were absent [3, 4]. Only a few of the Rac related GTPases have been characterized in detail. Rac1A, 1B and 1C [5, 6] and Rac E are required for cytokinesis , Rac1A was also shown to be involved in a formin-dependent pathway for filopodia formation , RacB is required for chemotaxis and morphogenesis  and RacC has been implicated in phagocytosis  and plays an important role in PI 3-kinase activation and WASP activation for the dynamic regulation of F-actin assembly during chemotaxis . RacG is required for cell shape, motility, and phagocytosis  and RacH has been implicated in vesicular trafficking .
Compared to our understanding of the downstream effects of Rac GTPases less is known about the mechanisms that activate Rac GTPases controlled by GEFs, GAPs or GDIs. In Dictyostelium at least 45 proteins contain a RhoGEF-PH module and most of them have a unique domain composition. The RhoGEF-PH (or diffuse B-cell lymphoma homology DH/pleckstrin homology PH) module is the structural feature that mediates the nucleotide exchange activity on Rho GTPases. Five of these RacGEFs have been studied in some detail. DdRacGap1 (DRG) containing both RhoGEF and Rho-GAP domains acts as a GEF for Rac1 and simultaneously acts as a GAP for RacE and Rab GTPases . RacGEF1 has a specificity for RacB in regulating chemoattractant stimulation, F-actin polymerization, and chemotaxis . The tail domain of MyoM, an unconventional myosin has been shown to catalyse nucleotide exchange on Rac1 GTPases and can induce actin-driven surface protrusions . More recently Trix, a three CH domain containing RacGEF, has been suggested to regulate the endocytic pathway . Finally, Darlin, an armadillo repeat protein homologous to the mammalian GEF smgGDS (small G-protein dissociation stimulator, a guanine nucleotide exchange factor for numerous Ras and Rho family GTPases ), has been shown to physically interact with RacE and RacC and may modulate chemotactic responses during early development . Nucleotide exchange activity on Rho GTPases are also displayed by CZH (CDM-zizimin homology) domain proteins . Presently only a few members of the family have been studied in other organisms like the mammalian Dock180 and CED-5 in C. elegans. Dictyostelium has 8 members of this family, but the functions of them remain to be elucidated.
In this study we focus on GxcDD, a novel multidomain RacGEF that contains a calponin homology (CH) domain, two IQ motifs, a DH domain, three PH domains and an ArfGAP domain. We show that, though dispensable for growth and development, GxcDD is required for proper streaming early in Dictyostelium development. The RacGEF domain of GxcDD can physically interact with several Rac GTPases. Characterization of the individual domains revealed that the CH domain can act as a membrane anchor and the ArfGAP-PH tandem accumulates at cortical regions and on phagosomes. Our data also suggest that a conformational change is possibly required to activate GxcDD.
Expression pattern and domain characterization of GxcDD
We analysed the expression profile of GxcDD during Dictyostelium development at the transcript level with a specific cDNA probe and at the protein level with polyclonal antibodies, respectively. We found that GxcDD is expressed throughout development and that the protein levels do not vary greatly (Fig. 1B, C). As the polyclonal antibodies that had been raised against the ArfGAP-PH domain of GxcDD, were unsuitable for immunofluorescence, we addressed the subcellular localization of the protein by means of subcellular fractionation and Triton X-100 treatment of the cells. We found GxcDD to be equally present in the cytosolic and membranous fractions. It also associated with the Triton X-100 insoluble cytoskeletal fraction (Fig. 1D).
The CH domain of GxcDD functions as a membrane association domain
Association of GxcDD with Dictyostelium Rac GTPases
The C terminal domain of GxcDD is enriched in the cortex and relocates to the membrane during phagocytosis
PH domains bind to phosphatidylinositol phosphates (PtdIns) and mediate the recruitment of proteins to membranes. When we tested whether the PH domain in the ArfGAP-PH domain could bind to PtdIns using a dot blot assay we found that the protein bound to PtdIns(3,4)P2 and PtdIns(4,5)P2, but highest binding was observed with PtdIns(3,4,5)P3, the product of PI3K (Fig. 5D). The PH-domain associated with the RacGEF domain could not be tested for binding to phosphatidylinositol phosphates so far as the protein was insoluble when expressed in E. coli.
Characterization of gxcDD- cells
gxcDD- cells did not show severely altered phenotypes when examined for growth on a bacterial lawn or in suspension, cytokinesis or actin organization in the cortex (not shown). The enrichment of the ArfGAP-PH domain at macropinosomes (Fig. 4A, B) suggested that GxcDD might have a role in endosomal processes like phagocytosis and pinocytosis. Quantitative phagocytosis and pinocytosis assays did however not reveal significant differences with the parental strain (not shown).
gxcDD- cells show a delay in development and defects in streaming behaviour
Analysis of cell motility in gxcDD- gells.
6.65 ± 2.61
5.53 ± 1.89
1.71 ± 1.19
1.38 ± 0.59
0.31 ± 0.19
0.32 ± 0.18
Direction change (deg)
47.81 ± 11.94
49.06 ± 14.28
14.56 ± 3.81
15.03 ± 3.75
5.55 ± 2.25
5.52 ± 2.27
0.71 ± 0.26
0.83 ± 0.14
Direction change (deg)
24.49 ± 17.76
17.32 ± 8.93
In the present study we have analysed GxcDD, a novel putative Dictyostelium RacGEF that has a unique domain organization containing a CH domain, two IQ motifs, a RhoGEF domain followed by two PH domains and an ArfGAP domain followed by one more PH domain. Seven other Dictyostelium RacGEFs contain a CH domain . The Calponin homology (CH) domain is a protein module of about 100 amino acids present in the actin-binding protein calponin that controls smooth muscle contraction and cytoskeletal organization in non-muscle cells. The CH domain of calponin is however not responsible for its interaction with actin . In contrast, the actin-binding domain (ABD) of several proteins is comprised of two CH domains in a tandem arrangement. CH domains are classified into at least five classes based on the degree of sequence similarity [22, 23]. CH1 in association with CH2 can interact with actin. Isolated CH2 and CH3 domains do not interact with actin at all [24, 25]. CH4 and CH5 are found in the actopaxin/parvin family of actin-binding proteins implicated in linking integrins with intracellular pathways that regulate the actin cytoskeleton . Most RhoGEFs from Dictyostelium and other species like mammalian Vav and α-PIX contain one type 3 CH domain and have been implicated in signal transduction. The CH domain of GxcDD is also a type 3 CH domain and our studies indicate that this domain is targeted to membranes exclusively and does not interact with actin. The membrane targeting of the CH domain may regulate the association of GxcDD with membranous fractions. CH3 domain containing proteins are the group of CH domain containing proteins that have the most diverse functions among the CH domains. Our findings are consistent with the hypothesis that although the five CH domains are homologous and have structural similarity, they may have evolved to perform different functions .
The DH domain of RhoGEFs regulates the nucleotide exchange activity. The associated PH domain is required for its full catalytic activity and for phosphoinositide binding and localization of the protein. Recent studies in yeast have shown that only a small fraction of PH domains are capable of independent membrane targeting of a protein and those that do often require phosphoinositides and non-phosphoinositide determinants like Arf GTPases in some cases for subcellular localization . GxcDD possesses a DH domain which is followed by two PH domains, the first being comparatively large and poorly conserved. We found that the DH domain can bind to a set of Rac GTPases (Rac1a, RacA, RacC, RacE, RacH and RacI) with sufficient affinity. However, our data need to be handled with caution, as mere physical interaction does not imply that all the above-mentioned Racs would be substrates for GxcDD in vivo. Further characterization of the interaction by enzymatic exchange assays needs to be performed for deeper understanding of its GEF activity. It is also possible that some of the interacting Racs and GxcDD might never see each other in vivo because of their subcellular localization e.g., RacH is mostly located at endosomes and not at the plasma membrane .
A conformational change or binding of bioactive lipids to proteins are mechanisms known to activate a signal transducing protein [29, 30]. Our observation of the ArfGAP-PH tandem binding to GxcDD but not being able to form higher oligomers indicates that the ArfGAP-PH may interact with some other part of the protein and exist as an inactive form. A conformational change would then be required to convert it to its active form. A possible activator could be PtdIns(3,4,5)P which binds to the ArfGAP-PH tandem with significant affinity. In an ArfGAP, ASAP1, a member of the centaurin family, which has an ArfGAP-PH tandem like GxcDD, the PH domain is known to function as an autoinhibitory domain and upon activation by PtdIns the ArfGAP domain is activated . A similar activation mechanism may also exist for GxcDD. The recruitment of the ArfGAP-PH tandem to the cortical regions of the cell and the phagocytic cup indicates a possible role in endocytic processes.
To understand the physiological role of GxcDD, we generated gxcDD- cells. Our observations indicate that although cells lacking GxcDD grow normally under laboratory conditions, undergo development and complete it with the formation of fruiting bodies, streaming behavior during chemotaxis is altered and development delayed. Dictyostelium cells can form streams during the aggregation process, which is the result of a cAMP relay mechanism. In this process certain cells, the pacemaker cells, have the ability to produce cAMP. The secreted cAMP can bind to cARs (cAMP receptors, which are G-protein coupled receptors), which induce a multitude of signaling events. Activation of cARs leads to dissociation of the heterotrimeric G-proteins and membrane localization of CRAC (c ytosolic r egulator of a denylyl c yclase), a PH domain containing protein, followed by activation of adenylyl cyclase (ACA), cAMP production and secretion . Activation of ACA is also regulated by another cytosolic regulator, Pianissimo . The neighboring cell then senses cAMP and streams of cells moving towards the aggregation centre are generated. A concerted action of 3-phosphoinositide metabolizing enzymes PI3K and PTEN are involved in tightly regulating the translocation of CRAC, Akt/PKB, PhdA (PH domain containg proteins) to the leading edge. Ras GTPases can activate PI3K. Ras C and its activator aimless RasGEF show decreased ACA activation. Cells lacking proteins that regulate ACA activity show defects in streaming or total loss and inability to aggregate or progress through development. It has been found that ACA localized to the rear of a migrating cell is required for formation of streams . ACA is possibly localized to the rear by a vesicular system that would require Arf function and GxcDD might be a part in its regulation. As GcxDD contains several functional and regulatory domains, it is likely to regulate signal transduction downstream of cAMP receptors (cARs). Binding of cAMP to the surface ligand activates PI3K whose products may use GxcDD as adaptor protein to bring about downstream signalling. GxcDD at the membrane may have either a function in conjunction with GTPases with its GEF and GAP domain or may act along with another combination of functional domains. Interference with such a complex network of intricate signalling is a possible mechanism for the delay in developmental timing in gxcDD- cells.
GxcDD is an unusual RacGEF containing CH domain, two IQ motifs, a RacGEF-PH tandem and an ArfGAP-PH tandem. It is present in cytosolic as well as membranous fractions. The CH domain of GxcDD localizes to the plasma membrane and may recruit GxcDD to the membrane and possibly regulate function and localization of the protein by binding to the ArfGAP-PH tandem. This is to the best of our knowledge the first evidence of membrane targeting of a CH, which would reveal an additional function for this functionally diverse domain. Our studies show that the RacGEF domain has the ability to physically interact with several Rac GTPases. To get a better insight to GxcDD function we need to perform nucleotide exchange assays to find the actual substrates for GxcDD. The ArfGAP-PH domain is associated with the leading edge of the cell and is able to associate with phosphatidyl inositides. It is also present in phagosomes and in Triton X-100 insoluble fractions. Deletion of the gene did not result in major phenotypic changes associated with growth, endocytic processes, actin organization, cytokinesis and cell motility, but shows subtle defects in formation of streams during aggregation and a delay in development.
Strain growth and development
D. discoideum cells of strain AX2 were grown either with Klebsiella aerogenes on SM agar plates or axenically in liquid nutrient medium  in shaking suspension at 160 rpm at 21°C. gxcDD- cells were cultivated in axenic medium containing 3.5 μg/ml blasticidin (ICN Biochemicals, OH). To analyse development, cells were grown axenically to a density of 2–3 × 106/ml, washed twice in Soerensen phosphate buffer (17 mM Na-K phosphate, pH 6.0) and 5 × 107 cells were plated on phosphate agar plates. The streaming pattern was studied by allowing 1 ml of 5 × 106 cells/ml to settle on a well of a NUNC six-well plate and observed at 3 min intervals with a Leica DM-IL inverse microscope.
Generation of gxcDD- cells and molecular cloning
For disruption of the GxcDD gene in AX2 cells, a GxcDD gene replacement vector was constructed using the plasmid pBSBsrΔBam . A 1.1-kb 5' fragment coding for the CH domain and the IQ motifs was PCR amplified using the forward primer 5-ATGCAACCCAAAGATTATATG-3' and reverse primer 5-ACTATTGTAATGGATGAT-3' and a 1.0-kb 3' fragment was PCR amplified using forward primer 5'-TTAATGAGTTGTATGAGAAGA-3' and reverse primer 5'-TGTGCAGAATGTGGAGCATCA-3' from AX2 genomic DNA. The PCR products obtained were cloned into pGEM-T Easy cloning vector (Promega GmbH). The gene fragments were released and cloned into pBSBsrΔBam. The resulting replacement vector was linearised by digesting with PvuII and transformed into AX2 by electroporation. Transformants were selected in nutrient medium containing blasticidin (3.5 μg/ml). Independent clones were screened for the disruption of the GxcDD gene by PCR using genomic DNA, Southern blotting and western blot analysis. For Southern blot analysis a probe encompassing nucleotides 3807–4860 was used.
For expression of the CH domain of GxcDD fused to green fluorescent protein (GFP) at the N-terminus, a 0.4-kb fragment encoding the first 134 residues of GxcDD was amplified from AX2 cDNA and cloned into pBsr-GFP [37, 38]. A 1-kb fragment encoding residues 395–707 containing the RacGEF domain was amplified and cloned in pGEX-4T1 (Amersham Biosciences) for expression in E. coli. The C-terminal 1-kb fragment encoding residues 1269–1619 containing the ArfGAP-PH tandem was amplified and cloned into pBsr-GFP for expression in AX2 cells and into pGEX-4T3 for expression in E. coli. Generation of strains expressing RacF1, RacG, RacH fused to GFP has been described elsewhere [12, 13, 39]. For expression of Rac1a, RacA (GTPase domain), RacB, RacC, RacD, RacE, RacI and RacJ fused to the C-terminus of GFP, PCR was performed on corresponding cDNAs. PCR products were cloned into pDEX-GFP  and the sequence verified. These vectors were introduced into AX2 cells and clones were selected by visual inspection under a fluorescent microscope.
Generation of polyclonal antibodies specific for GxcDD
Polyclonal antibodies specific for GxcDD were obtained by immunising female white New Zealand rabbits with GST-ArfGAP-PH (100 μg/animal; Pineda Antikörper-Service, Berlin), followed by two boosts of 100 μg each at two weeks intervals. The antiserum was affinity purified by incubating with GST-ArfGAP-PH bound glutathione-sepharose beads.
For separating membrane and cytosolic fractions cells were washed in Soerensen buffer and resuspended at a density of 1 × 108 cells/ml in MES buffer (20 mM MES, pH 6.5, 1 mM EDTA, 250 mM sucrose supplemented with protease inhibitor cocktail (Sigma)). Cells were lysed by sonication and membrane and cytosolic fractions separated by centrifugation at 100,000 g for 30 min at 4°C.
Triton X-100 was used for preparing cytoskeletal fractions. Cells were washed as before and resuspended in Soerensen buffer at a density of 5 × 107 cells/ml and 300 μl of cell suspension were lysed using an equal volume of TIC buffer (2% Triton X-100, 20 mM KCl, 20 mM imidazol, pH 7.0, 20 mM EGTA, 4 mM NaN3) and incubated on ice for 10 min followed by incubation at RT for 10 min. The insoluble cytoskeleton fraction was separated by centrifugation at 10,000 g for 4 min. Supernatant and pellet fractions were subjected to western blot analysis.
GST pulldown assays
GST-RacGEF and GST-ArfGAP-PH were expressed in E. coli and bound to glutathione-sepharose beads. For interaction of GxcDD with Rac proteins, 4 × 107 AX2 cells expressing Dictyostelium Racs as GFP fusions were lysed in lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 1 mM NaF, 0.5 mM Na3VO4, 1 mM DTT, supplemented with protease inhibitors (Sigma)) and incubated with equal amounts of GST-RacGEF bound beads for 3 hrs at 4°C. Beads were washed with wash buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA). The eluate of the pulldown was immunoblotted and the Rac protein detected using a GFP specific monoclonal antibody . Cells expressing only GFP were used as a control. For interaction studies of the ArfGAP-PH domain GST-ArfGAP-PH bound to glutathione-sepharose beads was incubated with AX2 cell lysates. For interaction studies of the CH domain with RacGEF and ArfGAP-PH domain, glutathione-sepharose beads bound to GST fused RacGEF and ArfGAP-PH tandem were incubated with AX2 cells expressing GFP-CH domain. Pulldown eluates were immunoblotted and probed with GFP specific monoclonal antibody. GST bound beads were used as a control.
Immunoflorescence was done by fixing cells using cold methanol for 10 min followed by staining for actin using actin-specific monoclonal antibody Act1-7  and Cy3 conjugated secondary antibody. Live cell imaging of fluorescent cells in suspension or during phagocytosis of TRITC labelled yeast particles was done by laser scanning confocal microscopy essentially as described . Capillary chemotaxis was done as described using a Leica DM-IL inverse microscope and analyzed using DIAS .
F-actin levels upon cAMP stimulation were determined as described . Briefly, aggregation competent cells resuspended at 2 × 107 cells/ml were stimulated with 1 μM cAMP and 50 μl samples were taken at various time points. Samples were immediately lysed in lysis buffer (3.7% formaldehyde, 0.1% Triton X-100, 0.25 μM TRITC-phalloidin in 20 mM potassium phosphate, 10 mM PIPES, pH 6.8, 5 mM EGTA, 2 mM MgCl2) and stained for 1 hr and centrifuged at 10,000 g for 5 min. Pellets were extracted with 1 ml methanol overnight and fluorescence (540/565) measured in a fluorimeter.
For crosslinking experiments GST-ArfGAP-PH was thrombin cleaved on glutathione-sepharose beads and the purified protein subjected to dialysis against PBS, pH 7.4, for 6 hrs at 4°C. To equal amount of protein increasing amount of glutaraldehyde (0–0.1% v/v) was added. The final reaction volume was 40 μl. Crosslinking was carried out at 4°C for 30 min. The reaction was stopped by addition of 5 μl 1 M glycine. Samples were subjected to imunoblotting.
Phosphoinositides binding was performed by a dot blot assay. 200 pmoles of each phophoinositide (PtdIns(3,4)P2, PtdIns(4,5)P2, PtdIns(3,4,5)P3) were spotted on a PVDF membrane and incubated with ArfGAP-PH domain. Protein bound to lipids was detected using polyclonal GxcDD antibodies.
For cell aggregation in suspension gxcDD- and AX2 cells were allowed to starve in Soerensen buffer at a density of 1 × 107 cells/ml and samples were withdrawn at the indicated times. The percentage of aggregated cells was determined by measuring the OD600.
For northern blot analysis total RNA was isolated from AX2 cells of different developmental stages as described previously and separated in agarose gels containing 6% formaldehyde [49, 50]. The blot was probed with a fragment derived from the 3' end of the GxcDD cDNA (nt 3807–4860)
We thank Rolf Mueller for technical help and advice and Berthold Gaßen for supplying monoclonal antibodies. The work was supported by the Deutsche Forschungsgemeinschaft funds NO 113/17-1 to AAN and RI 1034/4 to FR, the Fonds der Chemischen Industrie and the Köln Fortune Program of the Medical Faculty, University of Cologne.
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